Two-dimensional gel electrophoresis (2D-GE), since first published as a technique by O'Farrell (O'Farrell, P. H., J. Biol. Chem. 250:4007-21, 1975), has been for nearly 30 years the work horse in proteomics analysis, documented by thousands of application papers, and the object of numerous development and optimization attempts. Improvements occurred indeed and although alternative techniques, based on a different approach such as multidimensional chromatography directly coupled to mass spectrometry, are gaining popularity, 2D-GE is in fact still the most used technique and it might be still for several years if further improvements are achieved. Describing the details and advantages of 2D-GE in all its various forms is out of the scope of this invention as several reviews can be retrieved in the literature. It is however important to point out what the limitations of the general method still are and for this a general description of the process is only given.
Briefly the first dimension separation consists of isoelectric focusing (IEF) where proteins separate according to their isoelectric points in a pH gradient, typically immobilized (IPG), in a long and narrow supported gel assuming the form and taking the name of strip. The strips, commercially available, are normally supplied in a semi-dry state and they have to be rehydrated with the sample solution before analysis. This operation takes from a few hours to typically overnight and usually takes place under mineral oil to prevent drying and crystallization of urea present in the sample solution. IEF takes place also under mineral oil for the same reasons in the same or a different tray with the strip in contact with two electrodes at the sides, between which a high voltage is applied. After IEF the strip has to be equilibrated, which means that the proteins focused within the strip have to be first alkylated and then complexed with sodium dodecyl sulfate (SDS) in order to be later transferred to the second dimension gel and separated according to size. Reduction/alkylation can be achieved by different reagents and one has the option to perform this step during sample preparation before rehydration. It is actually recommended to do so (Herbert et al., Electrophoresis 22:2046-2057, 2001). However SDS equilibration can be performed only after IEF, so that the strip is literally washed for several minutes in the equilibration solution containing SDS. This is then placed on top and in contact with a prepolymerized SDS polyacrylamide gel and coupling is normally achieved by pouring a hot agarose solution over the strip. This is usually accomplished between two glass plates which are clamped together and then placed in a buffer containing cassette where voltage is applied across the gel. The gel might be formed with a porosity gradient in order to increase resolution in the second dimension. After this is complete, the gel is removed, fixed, stained and background staining dye removed before proceeding eventually with the subsequent steps, i.e. spot picking, digesting and mass spectrometry analysis.
All this is not only a time-consuming and laborious procedure, requiring trained personnel, but just because there is much manual work involved, reproducibility is still the major issue, as gels are mostly made to be compared. Although running conditions can be quite reproducible as these are controlled by proper set-up and power supplies, especially if fresh buffers are used all the time, problems with accuracy and consistency can arise from variations in the other numerous parameters to keep under control. For example, storage time (degradation) of precast gels prior to use, sample loading and rehydration, in terms of sample amount, losses, and homogeneity of the strip, strip handling with risk of damaging and contamination, equilibration between first and second dimension with risk of loosing sample and resolution, imprecise coupling to the gel, gel casting and polymerization, in terms of homogeneity, air sensitivity and risk to trap bubbles causing consequently also field inhomogeneities.
Ideally, what is desirable is that no further manual intervention is required after the sample has been loaded and that the overall process time is also reduced with the possibility to run several gels in parallel and increase not only the reproducibility but also the throughput while reducing the cost per gel. Automation and integration of the steps of this complex procedure is a challenge that others have already faced. Approaches making use of channels for separations instead of gels are not considered here as they are not directly comparable, neither are their performances, and because different parameters and limitations come into play.
An integrated, fully automated, system mimicking step by step the manual procedure, including also sample preparation and strip casting is described in U.S. Pat. No. 6,554,991 B1. The robotic machinery behind it, the complexity of the operation and the investment necessary go however far beyond a practical and widespread use of it, especially among the smaller research laboratories. US 2003/0127331 discloses a system where the strip once cast at the bottom of a vertical mold formed by two plates doesn't have to be moved after IEF. It is understood that the strip can be treated with the equilibration solution, apparently just from one side, and subsequently coupled to the second dimension gel by pouring the gel solution into the mold directly in contact with the strip or on top of an agarose layer. Doubts remain however concerning the efficiency and/or the time of the equilibration with the SDS having to diffuse inside the strip just from one side and whether the resolution obtained in the first dimension can be preserved. As no mention is made concerning the polymerization method, the long times associated with the classical method increase further the concern about loss of resolution. Also, the way the strip is formed and the sample is added is less reproducible and the fact that a sealing tab at the bottom of the mold has to be removed at the end is not practical. In EP 0366897 it is proposed that the strip is first separated from a prepolymerized gel by means of a non-conductive phase-change material, which is melted after IEF by increasing the temperature, removed and substituted with other gel medium. No reference is however made to the equilibration step and besides the concerns about the effect of the temperature for proteins and gel, remains the problem associated with closing and opening this time the top of the mold. Other barrier means between strip and gel are proposed in WO 02/084273 A1. The first embodiment reported therein making use of sliding solid barriers is certainly not the most advantageous as formed gels might be disrupted by this action. More interesting solutions make use of pneumatically assisted valves consisting of soft and expandable material or of semi-walls at the sides of the strip, which is physically bound as well as the gel on a flexible and peelable foil. This foil can change position relative to the opposite rigid surface, thus opening and closing the strip chamber. The gel solution is in this case introduced and polymerized after opening the strip chamber at the end of the first dimension. Equilibration solutions can access the strip through slits correspondingly positioned on the rigid part of the device. An automated system eventually based on this principle will be commercialized by Nextgen Sciences in the near future. This however reproduces also the same steps which are otherwise carried out manually, thus demanding a certain complexity due to valves and moving parts and adding cost for the instrument, also this less affordable. Other weak points of this system are represented by dead volumes for the sample, certain loss of resolution due to the equilibration between first and second dimension and the long waiting time for gel polymerization. Within the U.S. Pat. No. 6,277,259 an automated two dimensional gel electrophoresis of proteins is described, using thin linear gels. The protein sample is dissolved and complexed and processed in a first dimension separation followed by migration into a second dimension separation, labelled by a dye e.g. a fluorescent labelling to enable detecting the proteinaceous sample during second dimension separation. Within the WO 03/092846 a two dimensional analysis in a micro fluidic format using microchannels is described for performing two dimensional separations.
A cheaper, simpler, quicker and equally integrated solution in the form of a disposable, specially designed for minigel format is offered by Roche Diagnostics as described in a previous not yet published EP application (EP 04 015 469.2 attached to this application).